Neurotransmitter-gated membrane ion channels have been found to be one of the major sites of alcohol action in the nervous system. However, our knowledge of the molecular mechanisms of neurotransmitter-gated membrane ion channel function is relatively limited. We are using a combination of molecular biological and electrophysiological techniques to elucidate the molecular mechanisms of neurotransmitter-gated membrane ion channel function and the interaction of alcohol and other neuroactive substances with those mechanisms. Gamma-aminobutyric acid type A (GABA-A) receptors are the major inhibitory neurotransmitter-gated membrane ion channels in the mammalian brain. GABA-A receptors consist of multiple subunits and exhibit distinct pharmacological and channel properties. Of all the GABA-A receptor subunits, the beta subunit is thought to be a key component for the functionality of these receptors. Certain types of GABA-A receptors have been found to open spontaneously. However, the molecular basis for the spontaneous opening of these receptor-channels is poorly understood. In this study, we found that channels that contained beta-1, but not beta-3, subunits opened spontaneously when these subunits were expressed homomerically or co-expressed with other types of GABA-A receptor subunits in Xenopus oocytes. Using subunit chimeras and site-directed mutagenesis, we localized a key amino acid residue, serine (S) at 265 [S265], that is critical for conferring spontaneous opening of beta-1 subunit-containing GABA-A receptors. In addition, some point-mutations of S265 also produced constitutively active channels. The magnitude of the spontaneous opening of these channels was found to be correlated with the molecular volume of the residue at 265 for both homomeric and heteromeric GABA-A receptors, suggesting that the spontaneous activity of beta-1 subunit-containing GABA-A receptors may be mediated through a molecular mechanism that is dependent on the molecular volume of the residue at 265. Experiments are also in progress to elucidate the molecular mechanisms involved in the function and the alcohol & neuroactive substance sensitivity of other neurotransmitter-gated membrane ion channels, such as N-methyl-D-aspartate (NMDA) receptors, nicotinic acetylcholine (nACh) receptors, glycine receptors and ATP receptors. These studies hold the promise that such molecular approaches will advance our knowledge of the molecular basis of alcohol and neuroactive substance action in the nervous system and provide a foundation for understanding the molecular basis of alcohol abuse and alcoholism.